Effect of Aloe vera whole leaf extract on short chain fatty acids production by Bacteroides fragilis, Bifidobacterium infantis and Eubacterium limosum

Aims: To investigate the effect of Aloe vera whole leaf extract on pure and mixed human gut bacterial cultures by assessing the bacterial growth and changes in the production of short chain fatty acids. Methods and Results: Bacteroides fragilis, Bifidobacterium infantis, and Eubacterium limosum were incubated with Aloe vera extracts [0%, 0·5%, 1%, 1·5% and 2%; (w/v)] for 24 and 48 h. Short chain fatty acids production was measured by gas chromatography/mass spectrometry analyses. A significant linear increase in growth response to Aloe vera supplementation was observed at 24 h for each of the bacterial cultures; however, only B. infantis and a mixed bacterial culture showed a significant positive linear dose response in growth at 48 h. In pure bacteria cultures, a significantly enhanced dose response to Aloe vera supplementation was observed in the production of acetic acid by B. infantis at 24 h and of butyric acid by E. limosum at 24 and 48 h. In the mixed bacterial culture, the production of propionic acid was reduced significantly at 24 and 48 h in a dose‐dependent fashion, whereas butyric acid production showed a significant linear increase. Conclusions: The results indicated that Aloe vera possessed bacteriogenic activity in vitro and altered the production of acetic, butyric and propionic acids by micro‐organisms selected for the study. Significance and Impact of the Study: The results of the study suggest that consumption of a dietary supplement, Aloe vera, may alter the production of short chain fatty acids by human intestinal microflora.     

Lipid-Enhanced Ethanol Production by Kluyveromyces fragilis

The fermentation ability of a strain of Kluyveromyces fragilis, already selected for rapid lactose-fermenting capability, was improved dramatically by the addition of unsaturated fatty acids and ergosterol to the medium. The fermentation time of a 20% whey-lactose medium was decreased from over 90 h to less than 60 h. The lipids were shown to be taken up by the organism, and the effects on specific growth rate and biomass production were determined.     

In Vitro Study and Comparison of Caecal Methanogenesis and Fermentation Pattern in the Brown Hare (Lepus europaeus) and Domestic Rabbit (Oryctolagus cuniculus)

The brown hare and the domestic rabbit are mid-sized herbivorous mammals and hindgut fermenters, though their digestive physiologies differ in some traits. The objective of this study was to estimate and compare the caecal microbial activity in hares and rabbits via an analysis of the following end-products of in vitro caecal fermentation: methane, total gas production, short chain fatty acids and ammonia concentration. Hare caecal methanogenesis occurred at a much lower level (0.25 mmol/kg for samples incubated without substrate and 0.22 mmol/kg for samples incubated with substrate) than that of the rabbit (15.49 and 11.73 mmol/kg, respectively) (P<0.001). The impact of the substrate’s presence on caecal methanogenesis was not significant, though its presence increased the total gas production during fermentation (P<0.001). Hare caecal microflora produced a lower short chain fatty acids concentration than did rabbit microorganisms (P<0.05). In unincubated hare samples, the short chain fatty acids concentration was 28.4 mmol/kg, whereas in unincubated rabbit samples, the short chain fatty acids concentration was 51.8 mmol/kg. The caecal fermentation pattern of the hare was characterised by higher propionate and isobutyrate molar proportions compared with those observed in rabbit caecum (P<0.01). No significant changes in the ammonia concentration in either rabbit or hare caecum were found. The results obtained indicate some differences in the activity of the microbial populations colonising the hare and rabbit caecum, particularly in regards to methanogenic Archaea.     

The expression of the Cuphea palustris thioesterase CpFatB2 in Yarrowia lipolytica triggers oleic acid accumulation

The conversion of industrial by‐products into high‐value added compounds is a challenging issue. Crude glycerol, a by‐product of the biodiesel production chain, could represent an alternative carbon source for the cultivation of oleaginous yeasts. Here, we developed five minimal synthetic glycerol‐based media, with different C/N ratios, and we analyzed the production of biomass and fatty acids by Yarrowia lipolytica Po1g strain. We identified two media at the expense of which Y. lipolytica was able to accumulate ～5 g L^?1 of biomass and 0.8 g L^?1 of fatty acids (0.16 g of fatty acids per g of dry weight). These optimized media contained 0.5 g L^?1 of urea or ammonium sulfate and 20 g L^?1 of glycerol, and were devoid of yeast extract. Moreover, Y. lipolytica was engineered by inserting the FatB2 gene, coding for the CpFatB2 thioesterase from Cuphea palustris, in order to modify the fatty acid composition towards the accumulation of medium‐chain fatty acids. Contrary to the expected, the expression of the heterologous gene increased the production of oleic acid, and concomitantly decreased the level of saturated fatty acids. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:26–35, 2016     

Release of short chain fatty acids from cream lipids by commercial lipases and esterases

Lipases and esterases are frequently used in dairy production processes to enhance the buttery flavour of the end product. Short chain fatty acids, and especially butanoic acid, play a key role in this and different enzymes with specificity towards short chain fatty acids are commercially available as potent flavouring tools. We have compared six lipases/esterases associated with buttery flavour production. Although specificity to short chain fatty acids was ascribed to each enzyme, clear differences in free fatty acid profiles were found when these enzymes were applied on cream. Candida cylindraceae lipase was the most useful enzyme for buttery flavour production in cream with the highest yield of free fatty acids (57 g oleic acid 100 g⁻¹ fat), no release of long chain fatty acids and specificity towards butanoic acid.     

灌喂植物乳杆菌和干酪乳杆菌增加仔猪肠道菌群多样性及短链脂肪酸生成

【目的】研究断奶前给仔猪饲喂植物乳杆菌和干酪乳杆菌对断奶前、后肠道菌群组成、数量和短链脂肪酸(SCFA)浓度的影响,分析仔猪生长性能与肠道形态、微生物菌群及SCFAs的相关性,探讨测试菌株缓解仔猪断奶应激的可能机制。【方法】选取15窝7 d龄杜长大仔猪,随机分为3组,分别灌喂2 mL去离子水(对照组)、0.5×10~9 CFU/mL植物乳杆菌(LP组)或干酪乳杆菌(LC组)的菌液,每组以窝为单位5个重复,于21 d(断奶)、24 d和35 d屠宰,采集回肠和结肠食糜,分析菌群组成和数量的变化,测定SCFAs浓度。【结果】测试菌株均能显著提高断奶2周后回肠、结肠菌群多样性(P0.05),促进乳酸杆菌和双歧杆菌增殖;显著促进断奶前回肠和结肠中乙酸、丙酸、丁酸和总SCFA生成,促进断奶后乙酸和总SCFA产生;相关分析显示,测试菌株组仔猪腹泻率下降与SCFAs浓度上升、回肠绒毛高度增加和总菌数量上升显著相关,日增重提高与结肠乙酸和TSCFA浓度增加显著相关。【结论】测试菌株促进乳酸杆菌、双歧杆菌等有益菌增殖,增加肠道菌群多样性,促进肠道SCFAs生成。     

Enhancement of fruity aroma production of Pseudomonas fragi grown on skim milk,whey and whey permeate supplemented with C3-C7 fatty acids

Summary Pseudomonas fragi strain CRDA 037 produced a fruity aroma when grown in skim milk-, whey-and whey permeate-based culture media. The production of the odour-active metabolites was related to the lipid content of these media but was not influenced by the pH of the cultures. Analysis of the fruity aroma revealed that esters of fatty acids were some of the odouractive metabolites. Addition of C₃-C₇ fatty acids to the culture at 0 h stimulated the production of the corresponding fatty acid esters from 12 to 1570 times compared to unsupplemented media. Supplementation of the culture media with the C₃-C₇ fatty acids at 48 h, resulted in a 1.4- to 932-fold increase in the ethyl ester concentration.     

Lipids of the antarctic sea ice diatom Nitzschia cylindrus

The sterol and neutral, glyco- and phospholipid fatty acid profiles of the sea ice diatom Nitzschia cylindrus, isolated from McMurdo Sound, Antarctica, are reported. Two sterols were detected, trans-22-dehydrocholesterol (66% of total sterols) and cholesterol (34%); no sterols containing alkyl groups at the C₂₄ position were present. The major fatty acids in N. cylindrus, 16:1Δ9c, 14:0, 16:0, 20:5Δ5,8,11,14,17 and 20:4Δ5,8,11,14, were typical of previous reports of diatom fatty acids. A number of long-chain monounsaturated fatty acids were also detected, with higher relative proportions present in the phospholipid fraction. GC-MS analysis of the dimethyldisulphide adducts of these monounsaturated components showed that 24: 1Δ13c, 24:1Δ15c, 26:1Δ15c and 26:1Δ17c were the major components. The distribution of these fatty acids suggests that chain elongation of monounsaturated fatty acids was occurring in N. cylindrus. The proposed chain lengthening occurring for N. cylindrus represents, to our knowledge, the first report of possible chain lengthening of monounsaturated fatty acids in microscopic algae. These features, the presence of long-chain monounsaturated fatty acids and the sterol profile, may allow the input of this alga into benthic marine sediments or food webs to be monitored.     

Biodegradation of C5-C8 fatty acids and production of aroma volatiles by Myroides sp. ZB35 isolated from activated sludge

In the effluents of a biologically treated wastewater from a heavy oil-refining plant, C₅-C₈ fatty acids including pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, and 2-methylbutanoic acid are often detected. As these residual fatty acids can cause further air and water pollution, a new Myroides isolate ZB35 from activated sludge was explored to degrade these C₅-C₈ fatty acids in this study. It was found that the biodegradation process involved a lag phase that became prolonged with increasing acyl chain length when the fatty acids were individually fed to this strain. However, when fed as a mixture, the ones with longer acyl chains were found to become more quickly assimilated. The branched 2-methylbutanoic acid was always the last one to be depleted among the five fatty acids under both conditions. Metabolite analysis revealed one possible origin of short chain fatty acids in the biologically treated wastewater. Aroma volatiles including 2-methylbutyl isovalerate, isoamyl 2-methylbutanoate, isoamyl isovalerate, and 2-methylbutyl 2-methylbutanoate were subsequently identified from ZB35 extracts, linking the source of the fruity odor to these esters excreted by Myroides species. To our best knowledge, this is the first finding of these aroma esters in bacteria. From a biotechnological viewpoint, this study has revealed the potential of Myroides species as a promising source of aroma esters attractive for food and fragrance industries.     

Fatty Acid Synthase Impacts the Pathobiology of Candida parapsilosis In Vitro and during Mammalian Infection

Cytosolic fungal fatty acid synthase is composed of two subunits α and β, which are encoded by Fas1 and Fas2 genes. In this study, the Fas2 genes of the human pathogen Candida parapsilosis were deleted using a modified SAT1 flipper technique. CpFas2 was essential in media lacking exogenous fatty acids and the growth of Fas2 disruptants (Fas2 KO) was regulated by the supplementation of different long chain fatty acids, such as myristic acid (14∶0), palmitic acid (16∶0), and Tween 80, in a dose-specific manner. Lipidomic analysis revealed that Fas2 KO cells were severely restricted in production of unsaturated fatty acids. The Fas2 KO strains were unable to form normal biofilms and were more efficiently killed by murine-like macrophages, J774.16, than the wild type, heterozygous and reconstituted strains. Furthermore, Fas2 KO yeast were significantly less virulent in a systemic murine infection model. The Fas2 KO cells were also hypersensitive to human serum, and inhibition of CpFas2 in WT C. parapsilosis by cerulenin significantly decreased fungal growth in human serum. This study demonstrates that CpFas2 is essential for C. parapsilosis growth in the absence of exogenous fatty acids, is involved in unsaturated fatty acid production, influences fungal virulence, and represents a promising antifungal drug target.     

A genomic view on syntrophic versus non-syntrophic lifestyle in anaerobic fatty acid degrading communities

In sulfate-reducing and methanogenic environments complex biopolymers are hydrolyzed and degraded by fermentative micro-organisms that produce hydrogen, carbon dioxide and short chain fatty acids. Degradation of short chain fatty acids can be coupled to methanogenesis or to sulfate-reduction. Here we study from a genome perspective why some of these micro-organisms are able to grow in syntrophy with methanogens and others are not. Bacterial strains were selected based on genome availability and upon their ability to grow on short chain fatty acids alone or in syntrophic association with methanogens. Systematic functional domain profiling allowed us to shed light on this fundamental and ecologically important question. Extra-cytoplasmic formate dehydrogenases (InterPro domain number; IPR006443), including their maturation protein FdhE (IPR024064 and IPR006452) is a typical difference between syntrophic and non-syntrophic butyrate and propionate degraders. Furthermore, two domains with a currently unknown function seem to be associated with the ability of syntrophic growth. One is putatively involved in capsule or biofilm production (IPR019079) and a second in cell division, shape-determination or sporulation (IPR018365). The sulfate-reducing bacteria Desulfobacterium autotrophicum HRM2, Desulfomonile tiedjei and Desulfosporosinus meridiei were never tested for syntrophic growth, but all crucial domains were found in their genomes, which suggests their possible ability to grow in syntrophic association with methanogens. In addition, profiling domains involved in electron transfer mechanisms revealed the important role of the Rnf-complex and the formate transporter in syntrophy, and indicate that DUF224 may have a role in electron transfer in bacteria other than Syntrophomonas wolfei as well. This article is a part of a Special Issue entitled: 18th European Bioenergetics Conference (Biochim. Biophys. Acta, Volume 1837, Issue 7, July 2014).     

Microbial conversion of distillery waste to bioenergy: Effects of media enrichment with low chain fatty acids and Candida sp.

During microbial methanogensis of diluted distillery waste or spent wash (initial COD 25,000 mg/l) in the presence of sodium acetate, sodium propionate, or sodium butyrate at the concentration of 2000 mg/l, biogas containing 22.0% to 39.4% methane was produced in 20 days in a semicontinuous fermentation system. The analysis of the volatile fatty acid spectrum of the effluent showed accumulation of 46.3% branched chain fatty acids. When the fermentation medium was supplemented with modified Smith and Mah (SM) medium containing electrolytes and 1% sodium acetate, production of methane went up to 59.0% in 18 days. With the addition of a strain of Candida sp. the coculture produced about the same volume of methane (61%) but the time required was reduced (14 days). COD (16600.8 mg/l) was reduced and the effluent contained only 3% branched chain fatty acids adn 96% straight chain fatty acids. Fortification with the sodium salts of three branched-chain fatty acids as sources of carbon in SM medium (alone) reduced methane production. Only 0.0% to 14.2% methane was recorded. By combining these acids with straight chain fatty acids in SM medium, methane production increased significantly (about 4-fold) in proportion to the concentration of straight chain fatty acids added.     

An integrated computational and experimental study for overproducing fatty acids in Escherichia coli

Increasing demands for petroleum have stimulated sustainable ways to produce chemicals and biofuels. Specifically, fatty acids of varying chain lengths (C₆–C₁₆) naturally synthesized in many organisms are promising starting points for the catalytic production of industrial chemicals and diesel-like biofuels. However, bio-production of fatty acids from plants and other microbial production hosts relies heavily on manipulating tightly regulated fatty acid biosynthetic pathways. In addition, precursors for fatty acids are used along other central metabolic pathways for the production of amino acids and biomass, which further complicates the engineering of microbial hosts for higher yields. Here, we demonstrate an iterative metabolic engineering effort that integrates computationally driven predictions and metabolic flux analysis techniques to meet this challenge. The OptForce procedure was used for suggesting and prioritizing genetic manipulations that overproduce fatty acids of different chain lengths from C₆ to C₁₆ starting with wild-type E. coli. We identified some common but mostly chain-specific genetic interventions alluding to the possibility of fine-tuning overproduction for specific fatty acid chain lengths. In accordance with the OptForce prioritization of interventions, fabZ and acyl-ACP thioesterase were upregulated and fadD was deleted to arrive at a strain that produces 1.70 g/L and 0.14 g fatty acid/g glucose (～39% maximum theoretical yield) of C_14–16 fatty acids in minimal M9 medium. These results highlight the benefit of using computational strain design and flux analysis tools in the design of recombinant strains of E. coli to produce free fatty acids.     

Effect of thiocarbamate herbicides on fatty acid synthesis by potato

S-ethyldipropylthiocarbamate (EPTC), S-(2,3-dichloroallyl)diisopropylthiocarbamate (diallate) and S-(2,3,3-trichloroallyl)diisopropylthiocarbamate (triallate) inhibited the formation of very long chain fatty acids by aged potato discs. Incorporation of acetate-[¹⁴C] into total fatty acids was inhibited 24% by EPTC, 50% by triallate and 55% by diallate at 10^?4 M. The relative sensitivity of very long chain fatty acid synthesis to thiocarbamates in potato tuber provides further evidence that these herbicides reduce cuticular wax by inhibiting fatty acid elongation.     

Use of formic acid to control vibriosis in shrimp aquaculture

Luminous vibriosis is a shrimp disease that causes major economic losses in shrimp industry as a result of massive shrimp kills due to bacterial infection caused by Vibrio species. Use of antibiotics to control Vibrio in shrimp aquaculture is not allowed in the United States and so it is necessary to develop an alternative pathogen control method for shrimp production. Short-chain fatty acids have been used as food preservatives for a long time. Organic acids are commonly added in feeds in animal production, such as chicken, pig, and cattle. In this study, growth inhibition effects of formic acid on five selected Vibrio species, namely Vibrio alginolyticus, Vibrio cholerae, Vibrio harveyi, Vibrio parahaemolyticus and Vibrio vulnificus were studied. The Vibrio bacteria were grown on both solid and liquid media using Muller-Hinton agar and alkaline peptone water, respectively, with various concentrations of formic acid. Bacterial growth was monitored in the liquid media using optical density method. The results showed significant inhibition of growth of all five Vibrio species by formic acid at low concentration. The effective concentration (EC₅₀) values were calculated for all five Vibrio species, which were less than 0.039% of formic acid. The results are encouraging to supplement formic acid in the shrimp feed as a control mechanism to reduce Vibrio outbreak in shrimp aquaculture system.     

Membrane engineering via trans unsaturated fatty acids production improves Escherichia coli robustness and production of biorenewables

Constructing microbial biocatalysts that produce biorenewables at economically viable yields and titers is often hampered by product toxicity. For production of short chain fatty acids, membrane damage is considered the primary mechanism of toxicity, particularly in regards to membrane integrity. Previous engineering efforts in Escherichia coli to increase membrane integrity, with the goal of increasing fatty acid tolerance and production, have had mixed results. Herein, a novel approach was used to reconstruct the E. coli membrane by enabling production of a novel membrane component. Specifically, trans unsaturated fatty acids (TUFA) were produced and incorporated into the membrane of E. coli MG1655 by expression of cis-trans isomerase (Cti) from Pseudomonas aeruginosa. While the engineered strain was found to have no increase in membrane integrity, a significant decrease in membrane fluidity was observed, meaning that membrane polarization and rigidity were increased by TUFA incorporation. As a result, tolerance to exogenously added octanoic acid and production of octanoic acid were both increased relative to the wild-type strain. This membrane engineering strategy to improve octanoic acid tolerance was found to require fine-tuning of TUFA abundance. Besides improving tolerance and production of carboxylic acids, TUFA production also enabled increased tolerance in E. coli to other bio-products, e.g. alcohols, organic acids, aromatic compounds, a variety of adverse industrial conditions, e.g. low pH, high temperature, and also elevated styrene production, another versatile bio-chemical product. TUFA permitted enhanced growth due to alleviation of bio–product toxicity, demonstrating the general effectiveness of this membrane engineering strategy towards improving strain robustness.     

Phospholipids of Entamoeba invadens.

The major phosphoglycerides present in Entamoeba invadens are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol. Furthermore, three different sphingolipids could be isolated from the amoeba. In addition to sphingomyelin and a phosphonolipid, ceramide phosphonylethanolamine, a previously unknown sphingolipid was present. This sphingolipid contained a long chain base, inositol, and phosphorus in the ratio of 0.97:0.97: 1.0 and could be identified as ceramide phosphorylinositol. The various individual phospholipids showed different rates of turnover. Phosphatidic acid and phosphatidylinositol had, relative to the other phospholipids, a short half-time of about 12 h. Phosphatidylethanolamine and ceramide phosphorylinositol had a half-time of about 24 and 30 h, respectively. The major phospholipid, phosphatidylcholine and also sphingomyelin and phosphatidylserine showed no turnover. In contrast to the phosphoglycerides, the sphingolipid composition of the amoeba cultivated in different media was rather variable, while the total sphingolipid content remained at 21% of the total amount of phospholipids. The amount of ceramide phosphorylinositol was almost doubled in the cells cultivated on the serum-free medium (T), whereas the amount of sphingomyelin and ceramide phosphonylethanolamine decreased. Evidence is presented that these alterations in the sphingolipid composition of E. invadens are related to the amount of unsaturated fatty acids which were present in the culture medium.     

An effective acid catalyst for biodiesel production from impure raw feedstocks

Biodiesel consists of fatty acids short chain alkyl esters produced through transesterification and esterification of fats and oils. Production of biodiesel is strongly affected by the purity of raw lipids, and catalysts play important role in these processes. Although direct utilization of impure feedstocks is more economical, their use necessitates development of effective catalysts to overcome hindering influences of impurities. In this study, sulfuryl chloride, thionyl chloride, acetyl chloride, p-toluenesulfonic acid, benzenesulfonic acid, methanesulfonic acid, dimethylsulfate and sulfuric acid were investigated as catalysts for the production of biodiesel because acids have higher tolerance to water and free fatty acids in oils and can simultaneously catalyze both the esterification and transesterification reactions. Sulfuryl chloride was found to be an effective catalyst for production of biodiesel from soybean oil, its waste oil and microalgal lipids.